Kristian K. Brandt, Mette H. Nicolaisen, Ole Nybroe, Jan Sørensen, Chris Rensing Section of Genetics and Microbiology Department of Plant and Environmental Sciences University of Copenhagen

MICROBIOLOGICAL AND MOLECULAR TOOLS FOR

SOIL REMEDIATION

1. Detailed characterization of biodegradation by in situ studies of the abundance, expression, and diversity of key catabolic genes in indigenous bacterial degraders

2. Designed reporter bacteria as toxicity and bioavailability sensors to monitor soil quality and contaminant bioavailability during soil clean-up operations

3. Pollution-induced community tolerance (PICT) and reverse PICT as sensitive and ecologically relevant soil quality indicators

Three proposed concepts and tools for microbe-assisted soil remediation

Case 1

Surveying abundance, expression and diversity of key catabolic genes

Catabolic gene (DNA) → Transcript (mRNA) → Protein (enzyme)

Surveying catabolic genes encoding key enzymes for biodegradation: Proof-of-concept with phenoxy acid model pesticides

2,4-D / MCPA

(R)-dichlorprop

(S)-dichlorprop

Downstream analysis Real-time PCR DGGE DNA sequencing

30 g sieved soil

(R,S)-dichlorprop herbicide

Trace amounts 14C-dichlorprop

NaOH trap

RNA/DNA co-extraction

Scintillation count Microcosms

Experimental set-up

Transcriptional analysis of rdpA ,sdpA and

tfdA genes from indigenous dichlorprop

degrader population

Direct substrate induction of rdpA and sdpA genes Expression of rdpA/sdpA is linked to the mineralization of dichlorprop tfdA genes also induced by dichlorprop

Paulin et al. (2011) Environ Microbiol 13: 1513–1523

Degrader community diversity studied by DGGE

analysis of catabolic genes in soil rdpA

sdpA

tfdA

Paulin et al. (2011) Environ Microbiol 13: 1513–1523

Identification of bacterial degraders based on - Sequencing of catabolic genes - Sequencing of actively

transcribed genes (mRNA)

D. Acidovorans MC1 Variovorax paradoxus S110

Class III

S. herbicidovorans MH

Current activities: Optimization of in situ biodegradation of hydrocarbons using alkB

gene (alkane degradation) as functional gene marker

• Alkane degrader community composition is affected by nutrient addition as determined by the use of alkB gene as functional gene marker

Bioaugmentation of hydrocarbon-

contaminated soils in biopiles

using patented system for

delivery of degrader organisms

(Cleanfield Denmark)

Anja Sandal Grønskov

Case 2

Designed reporter bacteria as toxicity and bioavailability sensors

Whole-cell bacterial biosensors (bioreporters) for quantification of bioavailable toxicants

Response to Cu by two recombinant Pseudomonas fluorescens DF57 biosensor strains

[CuSO4] µM

0.01 0.1 1 10

Bio

lum

inescence

(rela

tive lig

ht units)

0

200

400

600

800

1000

1200

1400

Bio

lum

inescence

(% o

f contr

ol)

0

20

40

60

80

100

120Strain Cu15

Strain 40E7

“lights off”

“lights on”

Analytical range

• A are anions: E.g. carbonate, phosphate, sulfide. • L are ligands: E.g. chloride, citrate, DOC (e.g. root

exudates). • Bio-available fraction: Cu2+ and some CuL ?

Cu2+ Cu CuL (aq)

CuA (s)

sorption

precipitation

complexation

Bioavailable fraction ?

Cu speciation and bioavailability in soil

Particle

Cu-EDTA complexes are NOT bioavailable

Impact of chemical speciation on bioavailability

Cu bioavailability in Cu-EDTA solutions

Nybroe et al. (2008) Environ Toxicol Chem 27: 2246–2252

Current activities: Spatial heterogeneity of bioavailable Cu as determined by bioluminescent bioreporter before and after soil remediation by soil washing technologies

See Brandt et al., 2008 (Environ Sci Technol 42:3102-3108) for details on bioreporter assay methodology.

Case 3

Pollution-induced community tolerance (PICT) and reverse PICT

Experimental field site (Hygum): Long-term (+85 yr) Cu gradient ranging from background Cu levels to high levels of Cu contamination (>3000 ppm)

High Cu soil

Pollution-induced community tolerance (PICT) constitutes an ecologically relevant soil quality indicator

Cu dose−response curves for the nonadapted bacterial community extracted from control soil (triangles) and the corresponding Cu-adapted bacterial community extracted from high-Cu soil (circles) as measured by the leucine incorporation method (left panel, n = 2) and the bromodeoxyuridine incorporation method (right panel, n = 3). Curves represent best fits using nonlinear logistic models.

Berg et al. Environ. Sci. Technol. 2010, 44, 8724-8728. DOI: 10.1021/es101798r Copyright © 2010 American Chemical Society

The pollution-induced community tolerance (PICT) concept: Cu exposure selects for Cu tolerance in soil bacterial communities

OTU, ≥97% similarity level 6538 random 16S rRNA gene sequences per sample

Current activites: • Reverse PICT: Does the bacterial community retain Cu

sensitivity after soil clean-up?

Current activites: • New PICT detection assays for organic priority pollutants and

for toxicant mixtures (metals and hydrocarbons) • Identifying toxic chemicals in field contaminated soils using

biosensors, PICT, comprehensive chemical profiling, and multivariate statistics

• WANTED: Complexly polluted field sites (metals plus hydrocarbons) that includes non-contaminated control soil

Jakub Modrzynski

Conclusions and perspectives

1. Catabolic gene approach holds promise for evaluation and optimization of biodegradation potential (activity, composition, and diversity of specific degrader community)

2. Several whole-cell bacterial biosensors are ready for environmental application (measures bioavailability or toxicity)

3. Pollution-induced community tolerance (PICT) indicates causal relationships between chemical exposure and effect, and reverse PICT holds promise for ecological validation of soil remediation

Pathways by which metal resistant N-fixing rhizobia can act as plant growth promoting bacteria in metal-impacted soils

Hao et al. (in revision) Int. J. Phytorem. Chris Rensing

Thanks for your attention

Villum Foundation

[3H]leucine incorporation assay for phenotypic community-level resistance profiling

DNA

Ribosomes

Synthesis of radiolabelled

proteins containing

incorporated [3H]leucine

Inner membrane Outer

membrane

[3H]leucine incorporation (3 hour incubation)

Protein synthesis

Biomass production

Bacterial growth

[3H]bromodeoxyuridine (BrdU) incorporation assay for phenotypic community-level resistance profiling

DNA

* *

Synthesis of radiolabelled

DNA containing

incorporated [3H]BrdU

Inner membrane Outer

membrane

* *

[3H]BrdU incorporation (3 hour incubation)

DNA synthesis

Cell division

Bacterial growth

Ribosomes

Experimental set-up

Downstream analysis Real-time PCR

RNA/DNA co-extraction

Addition of sorption site

competitor

water

• Frozen in liquid N2

• Freeze dried overnight

100 ppm

1000 ppm

10 000 ppm

Scintillation count Microcosms

Clay content

pH

Phosphate

The pollution-induced community tolerance (PICT) concept constitutes an ecologically relevant soil quality indicator

• PICT detection assays must be short (few hours) to eliminate selection during the PICT detection phase

Figure modified from Demoling & Bååth (2008)